Abnormal differentiation of superficial epithelia (e.g. the oral epithelium, epidermis) is an etiology shared among structural birth defects ranging from orofacial clefting to congenital ichthyosis. Linkage and association studies confirm that these genetic disorders have a genetic basis. However, in the case of orofacial clefting only about half of the heritable risk has been assigned to specific genes. Moreover, even in the case of ichthyosis, for which the causative genes of most of the common forms have been discovered, how the encoded proteins contribute to the differentiation of epidermis remains only poorly understood. Thus there remains a critical need to identify regulatory molecules that control the development of superficial epithelia, and to determine the interactions among them. Without a thorough understanding of the overall gene regulatory network responsible for the development of superficial epithelia, the efforts of genetic counselors wishing to predict risk for these disorders will continue to be severely limited. The zebrafish embryonic skin or periderm is a tractable model for superficial epithelia. Here we propose an in vivo, systems biology approach to deduce the architecture of the transcriptional network governing differentiation of zebrafish periderm. We will do so, in Aim 1, through extensive in vivo perturbation analyses, including epistasis experiments. Further, in Aim 2, with a combined approach using in vivo reporter studies and computational analyses we will identify cis-regulatory modules that control expression in the periderm. We will test the hypothesis that enhancers that are active at the same level within the network hierarchy will share a common organization. Our team includes an investigator with expertise in zebrafish embryology and one with expertise in genomic analysis of cis-regulatory modules in model systems. Successful completion of the proposed work will constitute the most exhaustive analysis of trans- and cis- acting elements driving differentiation of any vertebrate cell type conducted to date. Our expected outcome therefore is a dramatic advance in understanding the development of superficial epithelia, resulting in an improved ability to recognize sequence polymorphisms in coding and regulatory DNA and that are pathogenic for diseases of epithelial differentiation, which include disorders of skin and oro-facial clefting.